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Carcinogenicity

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Description of key information

The test article was not carcinogenic in mice and rats treated for 52 and 104 weeks, respectively, at a dose level of 1000 ppm in feed.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1968
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
presented study was performed prior to OECD 453
GLP compliance:
no
Remarks:
prior to GLP
Species:
rat
Strain:
other: Sprague Dawley, Caesarian derived strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, U.S.A.
- Age at study initiation: (P) approximately 5 wks
- Weight at study initiation: (P) Males: 83-98 g (92 g group mean bw); Females: 79-93 (86 g group mean bw)
- Fasting period before study: No data.
- Housing: Five to a cage (unless the number was reduced by mortality), in suspended cages fitted with wire-mesh floors.
- Diet: Sifted Spiller's Laboratory Small Animals Diet (autoclaved), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 5
- Air changes (per hr): No data.
- Photoperiod (hrs dark / hrs light): No data.
Route of administration:
oral: feed
Vehicle:
other:
Details on exposure:
Not further specified.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily (in diet)
Post exposure period:
None.
Dose / conc.:
1 000 ppm
No. of animals per sex per dose:
35 animals
Control animals:
yes, plain diet
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/MORTALITY: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: recorded initially and at weekly intervals throughout.
- From these individual figures were calculated the group mean body weights.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The quantity of food consumed by each cage of rats was recorded and the mean weekly intake calculated.
- The efficiency with which food was utilized was assessed by calculation of mean food conversion ratios (FCR values) during the period of fastest growth, as weights of food consumed per unit gains in body weight.

WATER CONSUMPTION: Yes
- Water consumption was measured daily over five days.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: after 26 weeks of tretment
- Dose groups that were examined: 3 females of testing group 2

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 104 weeks
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: 10 males and 10 females from groups 1 (control) and 2 (1000 ppm test substance)
- Parameters checked: packed cell volume, haemoglobin concentration, red cell count, white cell count, differential white cell count, mean corpuscular haemoglobin content (MCHC), mean cell volume (MCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 104 weeks
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: 10 males and 10 females from groups 1 (control) and 2 (1000 ppm test substance)
- Parameters checked: plasma urea, total plasma reducing substances, serum alkaline phosphatase, serum glutamate-pyruvate transaminase, serum electrolytes (sodium, potassium), total serum protein, albumin/globulin fractions

URINALYSIS: Yes
- Time schedule for collection of urine: After 102 weeks
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: pH, specific gravity, protein, reducing substances, glucose, ketones, bile pigments, bile salts, urobilin, blood pigments, microscopy of spun deposit

OTHER:
- Urine concentration test: During week 104, 5 males and 5 females from Groups 1 and 2 were subjected to a test designed to detect impairment of the urine-concentrating power of the kidneys.
Sacrifice and pathology:
SACRIFICE
- All rats killed in extremis, or found dead in the cage, were subjected to detailed macroscopic examination to locate the affected organ(s).
- Where practicable, a full spectrum of tissue samples was preserved in formaldehyde saline.
- All surviving rats were killed by exsanguination.

GROSS NECROPSY
- The macroscopic appearance of the tissues was noted and the weights of the following organs recorded: kidneys and liver
- Relative organ weights were calculated as percentages of bodyweight, x 100.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Sample tissues indicated below were preserved in 4% formaldehyde saline and prepared for microscopic examination:
duodenum, heart, ileum, jejunum, kidneys, liver, lungs, ovaries, spleen, stomach, testes, tumours

- Fixed tissue samples were embedded in paraffin wax; sections were cut at 5 µm and stained with haematoxylin and eosin.
- In addition, frozen sections of liver and kidney were cut at 12 µm and stained for fat with Oil RedO.
- In the first instance, microscopic examination was confined to:
# Rats that died.
# 10 males and 10 females of Groups 1 and 2 killed at 104 weeks.
# All rats in which tumours or abnormal growths had occurred.
# Eyes from rats displaying lens opacities at termination.
Statistics:
Student's 't' test employed to assess the significance of inter-group differences, where the data suggested evidence of a response to treatment.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
After 26 weeks, there were no apparent differences in general condition among the rats of the two groups. Apart from one abdominal abscess in a control female, and the presence of optic opacities in three treated females, no abnormalities have been recorded. After 76 weeks, there were no apparent differences in general condition among the rats of the two groups. After 104 weeks, there were no overt signs of response to treatment, and there were no differences between the groups in respect of general bodily condition.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no mortalities during the first 26 weeks. After 76 weeks, there were no apparent differences in general condition among the rats of the two groups. Fewer deaths have occurred in the treated males than in the untreated males. The greater incidence of mortality seen in the treated females (when compared to the untreated females) is due mainly to the sacrifice or death of animals bearing tumours; however, this does not represent a treatment-related finding. Neither the mortality rates throughout the experiment, the final mortality totals, nor the observations made post-mortem suggested any treatment-related difference between the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
After 26 weeks there appeared to be some slight depression of the bodyweight of treated males, whereas treated females appeared unaffected; after 52 weeks, a slight depression was present in both sexes.
At 76 weeks, treated females showed relatively greater depression of weight-gain than did the treated males. However, when examined statistically (Student's ' t ' test) , the weight-difference between treated and untreated females was significant only at the 10% level.
During the second half of the study, weight-gains in treated females were slightly less than those of control females; however, the differences in weight at 76 and 104 weeks were statistically significant only at the 40% level, and therefore should not be ascribed to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
After 26 weeks, a slightly higher food consumption has been recorded for treated females than for the control groupe females, and this i s also true of the males when assessed relative to bodyweight. The differences are, however, of little or no significance at present.
Up to 52 weeks, females receiving the test article consumed slightly more (103%) food than the untreated controls. Over the period week 62 to week 76, both groups ate closely comparable amounts of food. The males receiving the test item ate slightly less (99%) than the controls. These differences are of little or no significance in the present context.
After 104 weeks, the treatment with the test substance did not modify food intake in the testing group.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
After 26 weeks, food conversion ratios calculated for month 6 are elevated in both sexes of Group 2, and there is evidence of a trend towards this beginning at month 4.
After 104 weeks, FCR values, calculated for the period of fastest growth, revealed no evidence of any effect of the test substance.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In view of slightly increased urine output observed in Group 2 at 102 weeks, water consumption was also examined over a one-week period. Intake appeared slightly higher among treated animals.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 26 weeks of treatment, optic opacities were observed by gross inspection in three females of Group 2; these rats died before the terminal examination. At termination, more treated rats displayed lens opacities than did the untreated control rats; the difference was more notable in females. Opacities in control rats were mainly of the posterior, punctate type, whereas those in treated animals frequently extended along the posterior suture, finally presenting as a Y-shaped or diffuse trefoil opacity. Most of these eyes were examined histologically, but only senility changes were found.
Haematological findings:
no effects observed
Description (incidence and severity):
None of the parameters examined showed any treatment-related disturbance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No significant intergroup differences were discovered
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no evidence of renal malfunction in treated animals at termination. Slightly more urine was voided by both sexes of testing Group 2, but there was no suggestion of a frank polyuria. Urine concentration test: This test confirmed the conclusion drawn above. Urine concentrating capacity in treated rats was equivalent or superior to that of the untreated controls.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The livers of treated males were significantly heavier than those of the non-treated males (P<0.05 for absolute weights, P<0.01 for relative weights).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Age-associated pathology was evident in all animals. The body fat of the treated animals exhibited marked fluorescence under ultra-violet illumination.
A weak fluorescence was observed when the eyes were similarly exposed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Minimal myocardial fibrosis, observed in the control and testing group during the study (animals found dead/animals killed).
- Respiratory tract: In many animals there was a minimal degree of chronic interstitial pneumonitis. In the occasional animal this was associated with some collapse and bronchopneumonic consolidation. These inflammatory reactions were considered to be wholly unrelated to treatment.
- Cardiovascular system: In many animals there was evidence of some myocardial fibrosis, particularly in the left ventricle, occasionally associated with mineralization in some of the coronary vessels and in parts of the ascending aorta. These changes were not group specific, and were considered to represent an age-associated change.
- Liver: The condition of the hepatic architecture was considered in the majority of animals to be within normal limits. Some variation from normality was noted, particularly in relation to hepatocyte size, presence of fatty change, and some lymphocytic infiltration, seen in and around the portal tracts. In many animals evidence of some re-duplication of the bile-duct epithelium was noted. These changes were seen throughout both groups, and were considered in all probability to associate
with an age /nutritional complex, without prime toxicological significance.
- Kidney: A variable degree of progressive glomerulo-nephrosis was seen throughout the majority of animals examined. Progressive glomerulo-nephrosis denotes an increasing dilatation and atrophy of the tubules, associated with the presence of "colloid material". As this condition progresses it becomes associated with lymphocytic infiltration of the interstitial spaces, which show an increased fibroblastic response; in the most severe instances, glomerular lesions are seen associated with partdistortion and fatty change of the glomerular tufts. These changes, however, are known to be associated with an age/nutrition complex, and were not considered to be of any toxicological significance.
- Adrenal glands: Haemorrhagic degenerative cysts were noted, particularly in the females of both groups. This was considered to be an age-associated change.
- Eyes: Of 22 rats displaying lens changes at week 104, whose eyes were subsequently examined histologically, four showed swollen or distorted lens fibres; this total included one control animal. The few instances of lens-fibre distortion or retinal atrophy are attributed to normal ageing processes.
- Miscellaneous: Various other pathological entities were observed, and considered to be un-associated with treatment; these included peri-arteritis of the mesenteric and,testicular arteries (sometimes associated with atrophy of the seminiferous tubules), occasional ovarian cysts, and pyornetra. Keratitis or localized skin inflammatory reactions were noted in a few animals. The condition of the remainder of the tissues was considered to be within normal limits. It was concluded that there was no evidence of histological alteration ascribable to treatment.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No intergroup differences emerged in respect of tumour Incidence and, in the case of externally visible or palpable tumours, there were no significant differences in time of onset. See further information on results (table) below.
Description (incidence and severity):
REPRODUCTIVE STUDY:
After about one year of treatment, attempts were made to undertake a reproductive study, in which F1 and F2 generations would be examined in respect of mating performance, litter size, pup weight and growth, sex ratio and ano-genital distances. The F0 generation was too old for this to be completed, and it was abandoned after two pairings.
Dose descriptor:
NOEL
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No evidence to suggest that the test substance at 1000 ppm (formally equivalent to 50 mg/kg bw/d) in the diet might be carcinogenic.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No evidence of adverse reaction to treatment, deposition of fluorescent material constituted the sole treatment-related finding at a dosage of 1000 ppm (formally equivalent to 50 mg/kg bw/d).

Histopathology: Neoplastic

Males Females
Control Group Test group Control Group Test group
Subjected to histological examination (no.) 28 27 31 31
Pituitary tumour 4 5 13 14*
Mammary tumours: Fibro-adenoma and Adeno fibroma 0 3 26 16
Mammary tumours: Adenocarcinoma 0 0 1 2
Fibroma 1 3 0 1
Fibrosarcoma 1 1 0 2
Caecal tumour 0 0 0 1
Thyroid tumour 0 1 0 0
Parathyroid tumour 1 0 0 0
Testicular tumour 0 2 0 0
Adrenal tumour 0 2 0 1
Lymphosarcoma 1 0 0 0
Externally apparent tumours, not subjected to histological assessment due to advanced autolysis or canibalism 2 0 0 0

* includes one haemorrhagic mucoid pituitary that disintegrated during processing.

Executive summary:

The test article was fed continuously to rats for two years, at a concentration of 1000 ppm in the diet. A similar group of untreated rats was housed, fed and examined in exactly the same manner as these treated animals, except in that the test item was excluded from the diet. Treated animals remained indistinguishable from untreated controls in respect of growth performance, mortality rate and general condition; terminal haematology, biochemistry, urinalysis and histology revealed no evidence of any reaction to treatment. In particular, the total and differential tumour incidence was similar in the treated and untreated animals. By exposure to ultra-violet light, fluorescent material was demonstrable in body fat, and (to a much lesser extent) in the eyes of treated animals. Optic lens opacities of trefoil or Y-shaped configuration were evident in 10/36 treated rats surviving to termination. The incidence of these opacities was higher in the treated group than in the concurrent controls of this experiment, and indeed higher than that previously recorded among animals of the same age and strain. Whilst this might be suggestive of an effect of, the number of surviving animals in each group is small and this restriction possibly precludes the drawing of any hard and fast conclusion. The incidence of other types of opacities in this experiment was similar for both treated and control groups. In male rats only, a minor degree of liver enlargement was apparently associated with treatment. There was no evidence of altered liver histology, serum alkaline phosphatase activity or serum glutamatepyruvate transaminase activity in these animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data are also reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for carcinogenicity under Regulation (EC) No 1272/2008. 

Additional information

In an oral carcinogenicity study, male and female (35/sex) rats were fed continuously with the test substance at a dietary concentration of 1000 ppm over a period of 104 weeks. A group of rats fed the diet without test material served as control. Treated animals remained indistinguishable from untreated controls in respect of growth performance, mortality rate and general condition; terminal hematology, biochemistry, urinalysis and histology revealed no evidence of any reaction to treatment. In particular, the total and differential tumor incidence was similar in the treated and untreated animals. By exposure to ultra-violet light, fluorescent material was demonstrable in body fat, and (to a much lesser extent) in the eyes of treated animals. Optic lens opacities of trefoil or Y-shaped configuration were evident in 10/36 treated rats surviving to termination. The incidence of these opacities was higher in the treated group than in the concurrent controls of this experiment, and indeed higher than that previously recorded among animals of the same age and strain. Whilst this might be suggestive of an effect of the test substance, the number of surviving animals in each group is small and this restriction possibly precludes the drawing of any hard and fast conclusion. Most of these eyes were examined histologically, but only senility changes were found. The incidence of other types of opacities in this experiment was similar for both treated and control groups. In male rats only, a minor degree of liver enlargement was apparently associated with treatment. There was no evidence of altered liver histology, serum alkaline phosphatase activity or serum glutamate pyruvate transaminase activity in these animals. Based on the results, a NO(A)EL for carcinogenicity (and toxicity) of 1000 ppm was derived (Huntingdon, 1968).

In a second carcinogenicity study a dietary concentration of 1000 ppm was fed to mice (52 per sex and dose group) over a period of 52 weeks, followed by observation for a further 26 weeks (Huntingdon, 1969). 52 rats per sex fed diets without the test article served as control group. Throughout the investigation, mortality rates and prime causes remained grossly comparable for the treated and untreated groups. No treatment-related trends were seen in growth or food consumption. The examination of the treated mice under UV illumination revealed fluorescent deposits in adipose tissue. After 26 weeks withdrawal from treatment, fluorescence was somewhat reduced, although still evident. Furthermore, there was no evidence to suggest that the test substance administered at 1000 ppm in the diet, might be carcinogenic for mice; tumor incidence was unaffected by treatment with the test substance. It was concluded that there was no evidence of adverse reaction to treatment, and that deposition of fluorescent material constituted the sole treatment-related finding. Based on the presented results, a NO(A)EL for carcinogenicity (and toxicity) of 1000 ppm was derived.

Overall, there was no evidence of carcinogenic activity of the test substance.